The stereospecificity (enantiospecificity) is very important for the function of bioactive compounds, such as drugs, as only one of the enantiomers usually has the desired biological activity, while the other is inactive or even toxic. Therefore, a chemical synthesis of such molecules must involve a strenuous step of separating enantiomers at some stage of the process, or said synthesis must start with a single enantiomer of a chiral precursor (a chiral synthon). The use of enzymes in the synthesis of organic compounds, beside lowering the formation of byproducts and providing high reaction rates under mild reaction conditions, obviates the above mentioned predicaments of purely chemical synthesis, since the enzymatic reactions are regioselective and stereospecific. Many biotechnologies take advantage of biocatalysis, using either free enzymes or cells containing them.
Chiral α-hydroxyketones are versatile building blocks for the organic and pharmaceutical chemistry, e.g., for the synthesis of vitamin E, certain antifungals, antidiabetics, etc. One important chiral α-hydroxyketone is (R)-phenylacetyl carbinol ((R)-PAC), used as a synthon in the production of various drugs having α and β adrenergic properties, including L-ephedrine, pseudoephedrine, norephedrine, norpseudoephedrine, and phenylpropanolamine. These drugs are used as decongestants, antiasthmatics, vasoconstrictors, etc.
For many decades, (R)-PAC has been obtained by biotransformation of benzaldehyde using various species of fermenting yeast, mostly Saccharomyces cerevisiae (Scheme 1).

The activity of the enzyme pyruvate decarboxylase (PDC) is responsible for the formation of PAC in the yeast [Hanc O. et al., Naturwissenschaften 43 (1956) 498], in a synthetic side reaction accompanying the enzyme's normal decarboxylation of pyruvate to acetaldehyde.
Like other biotransformations using cells, the above process is limited by toxicity of benzaldehyde towards the yeast cells, and by formation of many by-products, for example benzyl alcohol, due to the action of different cellular enzymes. These factors reduce the yield of the target product, and complicate the purification procedure. Czech patent CS 93627 (1960) describes pretreating the yeast cells by strong acids to increase their resistance toward the reaction mixture before starting the biotransformation of molasses, crude sucrose, and benzaldehyde to PAC. East German patent DD 51651 (1966) describes dosing acetaldehyde together with benzaldehyde to a yeast fermentation broth to push the reaction to the direction of required products. WO 9004631 (1990) uses yeasts Saccharomyces cerevisiae or Candida flarei improved by mutagenesis in a biotransformation of benzaldehyde and pyruvate to PAC. The Japanese publication JP 09234090 (1997) describes the manufacture of (1R,2S)-1-phenyl-1,2-propanediol by a biotransformation of benzaldehyde and pyruvate using Saccharomyces cerevisiae. WO 9963103 (1999) relates to a biotransformation of a substituted aromatic aldehydes and pyruvate to the corresponding hydroxyl ketones, comprising yeast-mediated catalysis in organic solvents. Publication JP 2000093189 (2000) describes manufacturing optically active α-hydroxyketones using yeasts from the genera Torulopsis and Candida. In publication WO 0144486 (2001), substituted or unsubstituted aromatic aldehydes and pyruvate condense to produce carbinol compounds using yeast, in the presence of a supercritical liquid or a liquefied gas.
Application of pure enzymes as catalysts of the desired reaction has a potential to overcome some drawbacks of the whole-cell biotransformation. The synthetic potential of pyruvate decarboxylases (PDC) from S. cerevisiae and Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida has been investigated [E.g., Crout D. H. G. et al., Biocatalysis 9 (1994) 1–30]. When developing a reliable industrial process based on the purified enzymes for producing (R)-PAC, two factors are of primary importance—the efficiency of (R)-PAC formation and the stability of the enzyme under production conditions. Bruhn et al. [Eur. J. Biochem 234 (1995) 650–6; and DE 19523269 (1996)] improved the catalytic properties of PDC of Zymomonas mobilis by means of site-directed mutagenesis. However, the overall efficiency of the pyruvate utilization for the carboligation reaction remained very low, with only 3.5% of the pyruvate being converted to the desired product, and the bulk of pyruvate undergoing decarboxylation to acetaldehyde.
It is therefore an object of this invention to provide a biotransformation process for the preparation of chiral aromatic α-hydroxy ketones, including PAC, in a high yield from optionally substituted arylaldehydes and α-keto-acids. We have carried out systematic studies of acetohydroxyacid synthases (AHAS; belonging to the international classification group EC 4.1.3.18; known also as acetolactate synthase). The normal physiological reaction catalyzed by AHAS is decarboxylation-condensation of two α-keto acids (Scheme 2), producing an (S)-acetohydroxy acid, and requiring no additional driving force or redox agents. No regeneration of cofactors such as ATP or NAD are needed for the synthesis, only flavine adenine dinucleotide cofactor (FAD), thiamin pyrophosphate (TPP), and a divalent metal ion must be present.

We have discovered that AHAS enzymes can also utilize “unnatural” substrates [Barak Z. et al., J. Bacteriol. 169 (1987) 3750–6; Gollop N. et al., Biochemistry 28 (1989) 6310–7; and Ibdah M. et al., Biochemistry 35 (1996) 16282–91].
Another 2-hydroxy-3-oxoacid synthase, tartronate semialdehyde synthase (TSAS; belonging to the international classification group EC 4.1.1.47; known also as glyoxylate carboligase) is closely related to AHAS, not only by its catalytic activity, but also by its sequence, as well as by other properties. Its normal physiological reaction is also decarboxylation-condensation of two 2-oxoacids, and it usually produces tartronic acid semialdehyde from glyoxylic acid (Scheme 3).

We have found that TSAS can also utilize unnatural substrates as its reactants.
It is therefore a further object of this invention to provide a biotransformation process for preparing chiral aromatic α-hydroxy ketones, including PAC, from optionally substituted arylaldehydes and 2-oxoacids using a 2-hydroxy-3-oxoacid synthase related to the AHAS family.